Adjuvants are Key Factors for the Development of Future Vaccines: Lessons from the Finlay Adjuvant Platform

The development of effective vaccines against neglected diseases, especially those associated with poverty and social deprivation, is urgently needed. Modern vaccine technologies and a better understanding of the immune response have provided scientists with the tools for rational and safer design of subunit vaccines. Often, however, subunit vaccines do not elicit strong immune responses, highlighting the need to incorporate better adjuvants; this step therefore becomes a key factor for vaccine development. In this review we outline some key features of modern vaccinology that are linked with the development of better adjuvants. In line with the increased desire to obtain novel adjuvants for future vaccines, the Finlay Adjuvant Platform offers a novel approach for the development of new and effective adjuvants. The Finlay Adjuvants (AFs), AFPL (proteoliposome), and AFCo (cochleate), were initially designed for parenteral and mucosal applications, and constitute potent adjuvants for the induction of Th1 responses against several antigens. This review summarizes the status of the Finlay technology in producing promising adjuvants for unsolved-vaccine diseases including mucosal approaches and therapeutic vaccines. Ideas related to adjuvant classification, adjuvant selection, and their possible influence on innate recognition via multiple toll-like receptors are also discussed. © 2013 Pérez, Romeu, Cabrera, González, Batista-Duharte, Labrada, Pérez, Reyes, Ramírez, Sifontes, Fernández and Lastre

ELISA Cualitativo de IgA anti-Lipopolisacárido de Vibrio cholerae en Saliva de Humanos

An ELISA technique for IgA antibodies again V. cholerae lipopolisaccharide (LPS) in saliva was standardized. Humans volunteers were orally immunized with 0, 107, 108, 109 colony forming units of the candidate vaccine strain 638, El Tor Ogawa. Saliva samples were collected at 0, 7, 8, 9, 10 and 14 days after oral administration. Seroconversion was considered if IgA titers determined by optical density were higher than the cutoff value and if a two-fold increase of the initial titer was found. The technique was compared to the ELISPOT and to the Vibriocidal Antibody Assay and also the results obtained were compared to the experimental groups showing sensitivity of 90.3, 92.85, 93.3%; specificity of 86.9, 89.5, 96.0%; positive predictive value of 95.0, 96.2, 98.2%; negative predictive value of 77.0 y 79.2, 85.7% and efficiency of 89.4, 90.2, 94.1% respectively. The presence of specific IgA against LPS in saliva was demonstrated after immunization with the vaccine candidate. The results showed a kinetic profile with a maximum of IgA titter at day 9 post immunization with the highest frequency of positive titers found in the group immunized with 109 cfu.Se estandarizó un ELISA para detectar, en saliva, IgA contra el lipopolisacárido, principal antígeno protectogénico de Vibrio cholerae. Los voluntarios fueron inoculados por vía oral con dosis de 0, 107, 108, 109 unidades formadoras de colonias (ufc) del candidato vacunal El Tor Ogawa, cepa 638. Las muestras de saliva fueron tomadas seriadamente, a los 0, 7, 8, 9, 10 y 14 días postinoculación. Se consideró seroconversión si las densidades ópticas eran superiores al nivel de corte y si los incrementos en cualquier tiempo después de la inoculación duplicaban los valores iniciales. Los resultados del ELISA en saliva se compararon con: el ELISPOT, el Vobriocida y además con los grupos experimentales. Se obtuvieron sensibilidades de 90.3, 92.85 y 93.3%; especificidades de 86.9, 89.5 y 96.0%; 96.0%, valores predictivos positivos de 95.0, 96.2 y 98.2%, 98.2%; valores predictivos negativos de 77.0, 79.2 y 85.7% y eficiencias de 89.4, 90.2 y 94.1% , respectivamente. Se demostró la presencia de IgA anti LPS en saliva de los individuos inoculados con el candidato vacunal, así como una mayor concentración con el inoculo (109 ufc) y se obtuvo la máxima positividad a los 9 días

Standardization of Neisseria meningitidis Serogroup B Colorimetric Serum Bactericida Assay

The correlate of protection for serogroup B meningococci is not currently known, but for serogroup C it is believed to be the serum bactericidal assay (SBA). The current SBAs are labor intensive and the variations in protocols among different laboratories make interpretation of results difficult. A colorimetric SBA (cSBA), based on the ability of Neisseria meningitidis serogroup B to consume glucose, leading to acid production, was standardized by using group B strain Cu385-83 as the target. The cSBA results were compared to those obtained for a traditional colony-counting microassay (mSBA). Glucose and bromocresol purple pH indicator were added to the medium in order to estimate growth of cSBA target cell survivors through color change. Different variants of the assay parameters were optimized: growth of target cells (Mueller Hinton agar plates), target cell number (100 CFU/per well), and human complement source used at a final concentration of 25%. After the optimization, three other group B strains (H44/76, 490/91, and 511/91) were used as targets for the cSBA. The selection of the assay parameters and the standardization of cSBA were done with 13 sera from vaccinated volunteers. The titers were determined as the higher serum dilution that totally inhibited the bacterial growth marked by the color invariability of the pH indicator. This was detected visually as well as spectrophotometrically and was closely related to a significant difference in the growth of target cell survivors determined using Student’s t test. Intralaboratory reproducibility was ±1 dilution. The correlation between bactericidal median titers and specific immunoglobulin G serum concentration by enzyme immunoassay was high (r = 0.910, P < 0.01). The bactericidal titers generated by the cSBA and the mSBA were nearly identical, and there was a high correlation between the two assays (r = 0.974, P < 0.01). The standardized cSBA allows easy, fast, and efficient evaluation of samples

Mucosal and systemic immune responses induced by a Single Time Vaccination Strategy in mice

Vaccination is considered by World Health Organization as the most cost-effective strategy for controlling infectious diseases. In spite of great successes with vaccines, many infectious diseases are still leading killers, because of their inadequate coverage. Several factors have been responsible: number of doses, high vaccine reactogenicity, vaccine costs, vaccination policy, among others. Contradictorily, few vaccines are of single dose and even less of mucosal administration. However, more common infections occur via mucosa, where secretory immunoglobulin A plays an essential role. As an alternative, we proposed a novel protocol of vaccination called Single Time Vaccination Strategy (SinTimVaS) by immunizing two priming doses at same time, one by mucosal and one by parenteral routes. Here, the mucosal and systemic responses induced by Finlay Adjuvants (AF Proteoliposome (PL) 1 and AF Cochleate (Co) 1) implementing SinTimVaS in Balb/c mice were evaluated. One intranasal dose of AFCo1 and intramuscular dose of AFPL1 adsorbed onto Aluminum hydroxide, with BSA or TT as model antigens, administrated at the same time, induced potent specific mucosal and systemic immune responses. Also, we demonstrated that SinTimVaS using other mucosal routes like oral and sublingual, in combination with subcutaneous route elicits immune responses. SinTimVaS, as a new immunization strategy, could increase vaccination coverage and reduce time-cost vaccines campaigns, adding the benefits of immune response in mucosa.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Preparation of meningococcal group A Polysaccharide-tetanus toxoid conjugate and their immunogenicity in mice

El desarrollo de las vacunas conjugadas para Haemophilus influenzae tipo b, ha demostrado ser eficaz en infantes y en niños pequeños y ha sentado las bases para el desarrollo de vacunas conjugadas dirigidas contra otras enfermedades. En este trabajo nosotros describimos la obtención, por un nuevo mé todo, de un conjugado de polisacárido de meningococo grupo A (PsA) y toxoide tetánico (TT) como proteína portadora, así como la evaluación de la respuesta inmune de este conjugado en ratones Balb/c. Los grupos aminos generados por hidrólisis básica en el PsA fueron unidos a los grupos carboxilos del TT, por medio de la reacción con carbodiímida. El conjugado obtenido indujo una respuesta de IgG anti-PsA y de subclases de IgG (IgG1, IgG2a) anti-PsA en los sueros de ratones inmunizados. Además, se observó la producción de IFNγ por células de bazo de ratones inmunizados con conjugados. Estos resultados constituyen una evidencia del cambio de tipo de respuesta inmune, de timo-independiente a timo-dependiente, del polisacárido una vez conjugado.The development of polysaccharide-protein conjugate vaccines for Haemophilus influenzae type b, which have proven to be efficacious in infants and young children, has led to active development by a number of investigators of conjugate vaccines for other diseases. We describe here the obtention, by a new method, of a conjugate containing meningococcal group A polysaccharide (PsA) and tetanus toxoid (TT) as a carrier protein and the immune response evaluation in Balb/c mice. Amine groups generated by basic hydrolysis in PsA were successfully conjugated to carboxyl groups of tetanus toxoid (TT), using carbodiimide-mediated coupling. The conjugate induced anti-PsA IgG response and anti-PsA IgG subclasses (IgG1, IgG2a) in sera of immunized mice. In addition, the production of IFNγ by spleen cell of mice immunized with conjugate was observed. These results indicate that the conjugate changed the polysaccharide immune response from a thymus independent to a thymus dependent response.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Preparation of meningococcal group A Polysaccharide-tetanus toxoid conjugate and their immunogenicity in mice

El desarrollo de las vacunas conjugadas para Haemophilus influenzae tipo b, ha demostrado ser eficaz en infantes y en niños pequeños y ha sentado las bases para el desarrollo de vacunas conjugadas dirigidas contra otras enfermedades. En este trabajo nosotros describimos la obtención, por un nuevo mé todo, de un conjugado de polisacárido de meningococo grupo A (PsA) y toxoide tetánico (TT) como proteína portadora, así como la evaluación de la respuesta inmune de este conjugado en ratones Balb/c. Los grupos aminos generados por hidrólisis básica en el PsA fueron unidos a los grupos carboxilos del TT, por medio de la reacción con carbodiímida. El conjugado obtenido indujo una respuesta de IgG anti-PsA y de subclases de IgG (IgG1, IgG2a) anti-PsA en los sueros de ratones inmunizados. Además, se observó la producción de IFNγ por células de bazo de ratones inmunizados con conjugados. Estos resultados constituyen una evidencia del cambio de tipo de respuesta inmune, de timo-independiente a timo-dependiente, del polisacárido una vez conjugado.The development of polysaccharide-protein conjugate vaccines for Haemophilus influenzae type b, which have proven to be efficacious in infants and young children, has led to active development by a number of investigators of conjugate vaccines for other diseases. We describe here the obtention, by a new method, of a conjugate containing meningococcal group A polysaccharide (PsA) and tetanus toxoid (TT) as a carrier protein and the immune response evaluation in Balb/c mice. Amine groups generated by basic hydrolysis in PsA were successfully conjugated to carboxyl groups of tetanus toxoid (TT), using carbodiimide-mediated coupling. The conjugate induced anti-PsA IgG response and anti-PsA IgG subclasses (IgG1, IgG2a) in sera of immunized mice. In addition, the production of IFNγ by spleen cell of mice immunized with conjugate was observed. These results indicate that the conjugate changed the polysaccharide immune response from a thymus independent to a thymus dependent response.Colegio de Farmacéuticos de la Provincia de Buenos Aire

La gD2 coadministrada con el AFCo1 por vía intranasal induce inmunidad protectora contra virus de herpes simple tipo 2 en ratones

La infección por virus herpes simple tipo 2 (VHS-2) continúa siendo un problema de salud mundial. Esta infección es transmitida sexualmente y es la principal causa de úlceras genitales. La prevención de esta enfermedad requiere de la utilización de vacunas mucosales, pues las vacunas parenterales no han sido exitosas. Por otra parte, no existen adyuvantes mucosales, por lo que el desarrollo de estos es esencial para la estrategia de estas vacunas. La administración intranasal (IN) de la glicoproteína D del VHS-2 (gD2), coadministrada con el cocleato (AFCo1+gD2) sería igualmente efectiva con la gD2 incluida (AFCo1-gD2). Se inocularon ratones hembras C57BL/6 por la vía IN con gD2, contenida dentro del cocleato, coadministrada con el cocleato o gD2 sola. Se determinaron los niveles de IgG anti gD2 en suero y lavado vaginal, así como las subclases de IgG anti gD2 por ELISA. Se determinó la respuesta linfoproliferativa en células de bazo, el perfil de citoquinas Th1/Th2, los signos de la enfermedad y la protección frente al reto viral. Se observaron altos títulos de IgG e IgG2c anti gD2 en el suero de los animales inoculados con la gD2 y el AFCo1 como adyuvante. No se observaron diferencias significativas (p>0,05) entre los grupos que recibieron AFCo1+gD2 y los que recibieron AFCo1-gD2. Se observó un perfil de citoquinas tipo Th1 y un 100% de sobrevida en los grupos que recibieron el AFCo1 como adyuvante de la gD2, mientras que en el grupo que recibió la gD2 sola no se observó protección. Estos resultados indican que la gD2 puede ser utilizada coadministrada con AFCo1 por vía IN como un potencial candidato vacunal contra VHS-2

Respuesta inmune mucosal inducida por proteoliposoma y cocleato derivados de N. meningitidis serogrupo B

Mucosal vaccination offers attractive advantages to conventional systemic vaccination. Most pathogens enter or establish infection at mucosal surfaces. This represents an enormous challenge for vaccine development. Nevertheless, the availability of safe and effective adjuvants that function mucosally is the major limitation. Therefore, we investigated the impact of mucosal immunization with the Neisseria meningitidis B proteoliposome (AFPL1, Adjuvant Finlay Proteoliposome 1) and its-derived cochleate (Co, AFCo1). They contain multiple PAMPs as immunopotentiators and have delivery system ability as well as Th1 polarization activity. Groups of female mice were immunized by nasal, oral, intravaginal, or intramuscular routes with three doses with AFPL1/AFCo1 alone or containing ovalbumin or glycoprotein (g) D2 from Herpes Simplex Virus type 2 (HSV-2). High levels of specific IgG antibodies were detected in sera of mice vaccinated with either route. However, specific IgA antibodies were produced in saliva and vaginal wash only following mucosal delivering. The polarization to a Th1 pattern was confirmed by testing the induction of IgG2a/IgG2c antibody, positive delayed-type hypersensitivity reactions, and gIFN production. Additionally, AFCo1gD2 showed practically no vaginal HSV-2 replication and 100% protection against lethal vaginal HSV-2 challenge. In conclusion, the results support the use of AFCo1 as potent Th1 adjuvant for mucosal vaccines, particularly for nasal route